An Unbiased Evaluation of CK2 Inhibitors by Chemoproteomics

Abstract

Recently protein kinases have emerged as some of the most promising drug targets; and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/ threonine-protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. Elevated CK2 activity has been established in a number of cancers where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently the development of CK2 inhibitors has been ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz), and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) was carried out to elucidate the mechanism of action as well as inhibitor specificity of these compounds. Utilizing a chemoproteomics approach in conjunction with inhibitor-resistant mutant studies, CK2␣ and CK2␣ were identified as bona fide targets of TBB, TBBz, and DMAT in cells. However, inhibitor-specific cellular effects were observed indicating that the structurally related compounds had unique biological properties, suggesting differences in inhibitor specificity. Rescue experiments utilizing inhibitor-resistant CK2 mutants were unable to rescue the apoptosis associated with TBBz and DMAT treatment, suggesting the inhibitors had off-target effects. Exploitation of an unbiased chemoproteomics approach revealed a number of putative off-target inhibitor interactions, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme quinone reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors as well as drug specificity that will assist in the development of more specific next generation CK2 inhibitors.

A New View into the Regulation of Purine Metabolism: The Purinosome

“…As the concentration of TBB increased, purinosome dissociation was observed suggesting that TBB is acting on purinosome formation differently than the rest of the CK2 inhibitors. It is worth noting that TBB has been shown to be more selective for CK2 compared to DMAT and its parent compound, TBI, raising the question of whether off target (or indirect) effects of CK2 inhibitors are present and have a greater effect on purinosome formation than CK2 inhibition alone [49, 50]. …”

Unbiased Functional Proteomics Strategy for Protein Kinase Inhibitor Validation and Identification ofbona fideProtein Kinase Substrates: Application to Identification of EEF1D as a Substrate for CK2

“…In addition to their therapeutic potential, the development of many novel protein kinase inhibitors has yielded new opportunities to evaluate enzyme–substrate relationships for protein kinases and to further elucidate their regulatory participation in biological events. 10−14 However, to capitalize on the promise of protein kinase inhibitors for therapy and as tools to elucidate the role(s) of protein kinases in the regulation of biological processes, it is imperative that unbiased approaches be developed to evaluate inhibitor specificity(15) and to validate that inhibitors are effective when used in cells or in vivo .…”

“…43,44 Since many protein kinase inhibitors, including the majority of CK2 inhibitors, are ATP-competitive inhibitors, there is a clear need for methods that evaluate their specificity and the potential for off-target effects involving other members of the protein kinase family or other ATP-binding proteins that are abundant in human cells. 15,45 For example the CK2 catalytic site has unique features that can be exploited in the design of specific inhibitors and in devising strategies to evaluate the specificity of these inhibitors. Notably, CK2 is one of the few protein kinases with the ability to utilize GTP as a substrate(46) and the catalytic pocket that binds ATP/GTP is somewhat more compact than in other kinases because of the presence of bulky hydrophobic residues.…”

The cytosolic protein kinase CK2 phosphorylates cardiac calsequestrin in intact cells

“…Owing to our previous in vitro evidence implicating CK2 as CSQ2 kinase [18], we tested the effects of new competitive inhibitors of CK2 [36] on CSQ2 kinase activity in cell extracts. To allow us to compare effects of CK2-specific inhibitors, we first normalized endogenous CSQ2 kinase activities in myocytes, COS cells, and purified CK2 by comparing their relative ability to phosphorylate 2 μg pure CSQ2.…”